Presented by:Sharath .H.NM pharmacy

Dept of ph. analysis

Seminar on HPLC

Index

1.Liquid chromatography introduction

2.Types of hplc techniques

3.Hplc Instrumentation

Introduction

Liquid chromatography is the method, in which a dilute solution of sample is passed through a column packed with solid particles. Thus, liquid is passed through vertical columns under gravitational flow.•

The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett. His famous studies focused on separating compounds (leaf pigments), which were extracted from plants using a solvent.

During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography

However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. •

During this time, pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times of compounds being isolated by column chromatography • By the 1980's HPLC was commonly used for the

separation of chemical compounds. • New techniques improved separation, identification,

purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to immerge.

Advantages OF HPLC

• Separations is fast and have a high resolving power

• Continuous monitoring of the column effluent.• Can be applied to the separation and analysis

of very complex mixtures.• Repetitive and reproducible analysis using the

same column.••

• Adsorption, partition, ion exchange and exclusion column separations are applicable.

• Capable of handling macromolecules of high molecular weight.

• Suitable for pharmaceutical compounds.• Suitable for separating non volatile

substances.•

When a mixture of components is introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent travels slowest the component which has less affinity towards the stationary phase travels faster. • Since no two components have the same affinity

towards the stationary phase, the components are separated

PRINCIPLE OF SEPARATION IN HPLC

Types of HPLC techniques

1.Based on modes of chromatography

i) Normal phase mode.

ii) Reverse phase mode.

2.Based on principle of Chromatography.

i) Adsorption chromatography.

ii) Partition chromatography

iii) Ion exchange chromatography.

iv) Size exclusion/gel permeation chromatography.

v) Affinity chromatography.

vi) Chiral chromatography

•

3.Based on elution techniques.

i) Isocratic separation.

ii) Gradient separation

4.Based on the scale of operation

I) Analytical HPLC.

II) Preparative HPLC.

5.Based on the type of analysis.

I) Qualitative analysis.• ii) Quantitative analysis.

1.Based on modes of chromatography

Interaction or affinity between:

Polar-polar is more

Non polar-non polar is more where as the

Interaction or affinity between the polar- nonpolar is less.

Normal phase chromatography:

Separation of the polar analytes by partioning onto a polar stationary phase.

stationary phase- polar (silica gel, alumina)

mobile phase- non polar.•

Mechanism-interaction of stationary phase (polar surface) with polar components of the sample molecules.

Applications –Separation of non-ionic , non polar substances (Sio2).•

Reverse phase chromatography:

Separation of non polar analytes by partitioning onto a non polar stationary phase.

Mechanism-interaction of stationary phase non polar hydro carbon chain with non polar parts of the sample molecules.

Application – separation of non ionic and ion forming non polar to medium polar substances. (COOH –hydrocarbon).

•

2.Based on principle of chromatography

Adsorption chromatography

Usually polar stationary phase and non-polar mobile phase are employed.

Polar s.p – silica gel, alumina, porous glass bead.

Non polar m.p – heptane, benzene, acetone•

Partition chromatography

In this case liquid stationary phase is used.

Polar s.p - alcohol, methanol, water.

Non polar s.p – benzene, CHCl3,acetone.•

Ion exchange chromatography:

The principle involves reversible exchange of the functional groups.

Exchange of similar charged ion.

Stationary phases :

1) Cation exchangers.

2) Anion exchangers.

Mobile phase: Aqueous buffer system.

Applications: organic acids, organic bases, proteins, nucleic acid .•

Size exclusion chromatography:

Mixture of components with different molecular size are separated by gels.

Gel acts as a sieves which has crossed polymer linkage and hence a mixture of substances with different molecular sizes is separated.

• Mechanism by its diffusion effects

Soft gels like dextran, agarose or polyacrylamide are used.

Semi rigid gels like polystyrene, alkyl dextran in non aqueous medium are also used.

•

Affinity chromatography:

Affinity of the sample with specific stationary phases.

Technique widely used in field of Bio technology, Microbiology, Biochemistry.

3.Based on elution technique:

Isocratic elution.

Isocratic Elutions – Constant solvent composition, mobile phase

polarity stays constant throughout elution process. This is equivalent to isothermal separations in GC.

•

2) Gradient Elutions – Mobile phase composition (and thus polarityvaries throughout elution. This is

equivalent to temperatureprogramming in GC.

4.Based on the scale of operation

Analytical HPLC - For analytical purpose.

-No recovery of the sample.• -sample quantity very small (micro grams)

Preparative HPLC - For preparative samples.

-individual fractions of the sample collected using fraction collector.

-Collected samples can be re-used.• Ex: Separations of few grams of a mixture

5.Based on the type of analysis

Qualitative Analysis : To identify the compound and to detect the presence of any impurities.

Quantitative Analysis : To detect the amount of individual or several components in the mixture.

•

Principle of separation in HPLC:

-Mixture of components introduced into the column.

-These travel according to their relative affinities towards the stationary phase.

• - Component with more affinity towards stationary phase travel slower and Component with less affinity towards stationary phase travel faster.

STATIONARY PHASES (PACKING MATERIALS)

Stationary phases are the material consisting particles which are about 1 to 10 microns in average (often irregularly shaped).

Polar : Normal phase .

-Silica ,Alumina

-Cyano, amino or diol terminations on the bonded phase.

Non polar : Reversed Phase.

-C18 to about C8 terminals on the bonded phase.• -Also Phenyl and Cyano terminals.

Mobile Phases in the HPLC

Must do the following: - solublize the analyte molecules and the solvent they are in.

- be suitable for the analyte to transfer back and forth between during the separation process.

It must :

- be pure .

- be compatible with instrument.

- not interfere during detection.

- not dissolve the stationary phase.

- be readily available.• - Not be too compressible (causes pump /flow problems)

INSTUMENTATION OF HPLC

1) Degassing system2) Pump solvent delivery system3) Check valves4) Pulse damper5) Pre-columns6) Guard column7) Flow splitter8) Auto sampler9) Sample injection port10) Column

HPLC Instrumentation

DEGASSING SYSTEM

Sparging/Bubbling Vacuum filtration Ultra sonication

PUMP-SOLVENT DELIVERY SYSTEM The pumps are used to pass mobile phase through the column at high

pressure because the particles that are used pack HPLC columns are small enough ie: <50μm and also particle size of packing material is 5-10 μm

To prevent solvent flow from gravity pumps that develop pressure up to 5000psi are needed to force the mobile phase through column so that solvent stream enters the instrument at constant flow rate/pressure. In addition to this the pumps used in HPLC should have the following features

PUMPS1. Generation of pressure upto 5000psi2. Flow rate ranging from 0.1 to 100ml/min3. Flow control & flow reproducibility of plus

or minus 0.5%4. It should be composition resistant and give a

pulse free out putPumps are thus categorized into1.Mechanical pumps• Displacement pumps• Reciprocating pumps

2.Pneumatic pumps

MECHANICAL PUMPS

1. DISPLACEMENT PUMPS

WORKING

• Works on the principle of positive solvent pressure.

• Consist of screw or plunger which revolves continuously driven by motor.

• Rotatory motion provides continuous movement of the mobile phase which is propelled by the revolving screw at greater speed and pushes solvent through small needle like outlet.

• Consist of large syringe like chamber of capacity 250 – 500 ml.

ADVANTAGES

• Flow is pulse free.• Provide high pressure upto 200 – 475 atm. • Independent of column back pressure and viscosity of

solvent.• Simple operation.

DISADVENTAGE

• Limited solvent capacity • Gradient elution is not easy.

2. RECIPROCATING PUMPSWORKING• Pressure from a gas cylinder delivered

through a large piston drivers the mobile phase.

• Pressure on the solvent is proportional to the ratio of piston usually 50: 1.

3. CHECK VALVES They are used to control the flow of solvent

& back pressure

4. PULSE DAMPER Pulse are used to dampen or reduce the

pulses observed from the wavy baseline caused by pumps

PULSE DAMPER Important damping methods include• A triple headed pump: - two heads in different stages of

filling as the third is pumping.

• A tube with an air space or a flexible bellows or tube: Here a gas (air space) or a flexible metal vessel takes up some of the solution energy. When pump refills, this energy is released and a smooth pressure pulsation result.

• A restrictor: - In this method, a 25 cm length of 4 mm bore .stainless steel tubing. Packed with 20µm glass beds, is placed between the pump and the column.

5. PRE-COLUMNS A pre-column is packed with 37-53μm silica particles

(saturator column). It is being fitted between pump & the injector valve

ensures that the mobile phase is fully saturated with the silicate ions prior to the sample injection.

Thus its use reduces the adverse effects of low or high pH mobile phases which in turn substantially extend the life of the column. Their use is recommended in ion-exchange chromatography using buffered aqueous phase.

6. GUARD COLUMN • A short column placed between the sample injector and

the inlet of the main ("analytical") column• The guard column is packed with the same kind of

packing as the main analytical column, and is intended to absorb or pick up impurities in the sample or mobile phase that might damage the main column & increase the life time of main analytical column.

7. FLOW SPLITTER

• When a differential type of detector is used the flow of solvent is split just before it enters the sample injection port

• so that one portion directly goes to the reference side of the detector & a portion to the analytical column housed in a constant temperature chamber

8. AUTOSAMPLER• Here in this type of instruments their will be a piston metering

syringe type pump to suck the prestabilised sample volume into a line & than transfer it to the relatively large loop (approx 100ml)

• In a standard six port valve. The simplest Autosampler utilizes the special vial & displace the sample through the needle in to the valve loop.

• Most of the Autosamplers are microprocessor controlled & can serve as a master collector for the whole instrument

9. SAMPLE INJECTOR PORT

a. Septum injector b. Stop flow septum less injectionc. Rheodyne injectors

SEPTUM INJECTION PORT.

• Syringe is used to inject the sample through a self sealing inert septum directly into the mobile phase.

• Drawback: - leaching effect of the mobile phase with the septum resulting in the formation of ghost peaks.

STOP FLOW SEPTUMLESS INJECTION.

• Flow of mobile phase through the column is stopped for a while.

• Syringe is used to inject the sample.• Drawback: formation of ghost peak.

RHEODYNE INJECTORS• Operation of sample loop.– sampling mode– Injection mode.

• Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling valve, and subsequently injected into the mobile phase by suitable rotation

of the valve. Micro volume sampling valve operation of a Sampling loop.

10. Analytical Column

• Analytical column is most important part of the instrument dimensions of column are

• Column length: 5cm to 30cm• Column diameter: 2mm to 50mm• Particle size: 1μ to 20 μ• Particle nature: Spherical, uniform sized,

porous materials are used

Few Analytical columns

CLASSIFACTION OF CLOUMN BASED ON APPLICATION

Standard column

Packed column

Narrow bore column

Short fast column

Preparative columnMicro preparative columnPreparative columnMacro preparative column

STANDARD COLUMN • Internal diameter 4 – 5 mm and length 10 – 30 cm. • Size of stationary phase is 3 – 5 µm in diameter.• Used for the estimation of drugs, metabolites,

pharmaceutical preparation and body fluids like plasma.

NARROW BORE COLUMN

• Internal diameter is 2 – 4 mm. ( signal is increased 4 times )

• Require high pressure to propel mobile phase.• Used for the high resolution analytical work of compounds

with very high Rt

SHORT FAST COLUMN

• Length of column is 3 – 6 cm.• Used for the substances which have good affinity

towards the stationary phase. • Analysis time is also less (1- 4 min for gradient

elusion & 15 – 120 sec for isocratic elusion).

PREPARATIVE COLUMN

• Used for analytical separation i.e. to isolate or purify sample in the range of 10-100 mg form complex mixture.

Length – 25- 100 cmInternal diameter – 6 mm or more.

Preparative column are of three type :

• Micro preparative or semi preparative column

• Modified version of analytical column• Uses same packaging and meant for purifying sample less then 100 mg.

Preparative column

• Inner diameter – 25 mm .• Stationary phase diameter – 15- 100 µm

Macro Preparative Column

• Column length – 20 – 30 cm• Inner diameter – 600 mm

PACKING OF COLUMN• The most widely used method of packing column is by high pressure

slurring technique.• A suspension of packing is made in a solvent of equal density to the

packing material. • The slurry is then rapidly pumped at high pressure onto a column

with a porous plug at its outlet. • The resulting bed of packed material with in the column can then be

prepared for use by running the developing solvent through the column, hence equilibrating the packing with the developing solvent.

• When hard gels are packed, it is necessary for them to be allowed to swell first in the solvent to be used in the chromatographic process before packing under pressure.

• Soft gels cannot be packed under pressure and have to be allowed to pack from a slurry in the column under gravitational sedimentation only, in a similar way of packing conventional column

METHOD OF PACKING• Depends on the mechanical strength of stationary phase.• Particle size of the stationary phase.

– Particles of greater then 20 µm – dry packing– Particles of lesser then 20 µm – slurry packing / wet

packing.DRY PACKING

• Particle size greater then 20 µm filled into vertical clamped column in small quantity.

• Deposition is done by tapping or vibrating the column.

• Column is unclamped and the tapped on the firm surface to obtain dense and reproducible packing

WET / SLURRY PACKING• Particle size with diameter less then 20 µm

can only be placed wet as a suspension.• Suspension should be stable, it should not

sediment, and agglomentation should be avoided.

DETECTORS

Criteria• Selectivity• Sensitivity and detection limit• Stability• Reproducibility• Economically affordable• It should only record the component of interest

UV absorption detectors

• Fixed wavelength detectors• Multi wavelength detectors• Photo diode array detectors

Fixed wavelength detectors

Multiple wavelength detectors

Diode array detector• Here broad emission source like deuterium lamp is

collimated by an achromatic lens• Sample is subjected to all wavelength generated by

the lamp• Dispersed light from gratings allowed to fall on to

diode array• Array contain hundreds of diodes, output from each

diode is sampled by a computer

Photo diode array detector

FLUORESCENCE DETECTOR• The single wavelength excitation fluorescence detector is

probably the most sensitive LC detector that is available, but is achieved by forfeiting versatility. A diagram of a simple form of the fluorescence detector is shown in figure.

• The excitation light is normally provided by a low pressure mercury lamp which is comparatively inexpensive and provides relatively high intensity UV light at 253.7 nm. Many substances that fluoresce will be excited by light of this wavelength

• The excitation light is focused by a quartz lens through the cell. A second lens, set normal to the incident light, focuses the fluorescent light onto a photo cell. A fixed wavelength fluorescence detector will have a sensitivity (minimum detectable concentration at an excitation wavelength of 254 nm) of about 1 x 10-9 g/ml and a linear dynamic range of about 500 with a response index of 0.96 < r <1.04.

Flourosence detectors

ELECTROCHEMICAL DETECTORS

This detector is based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification

• The eluent should contain electrolyte and be electrically conductive. Most of the analytes to be successfully detected require the pH adjustments.

• The areas of application of electrochemical detection are not large, but the compounds for which it does apply, represent some of the most important drug such as phenol, catecholamines, nitrosamines, and organic acids are in the picomole (nanogram) range

• The specificity, and sensitivity make it very useful for monitoring these compounds in complex matrices such as body fluids and natural products.

Deflection detectors • The optical schematic of the deflection detector is shown

in below. This detector based on the deflection principle of refractometry

• where the deflection of a light beam is changed when the composition in the sample flow-cell changes in relation to the reference side (as eluting sample moves through the system).

• When no sample is present in the cell, the light passing through both sides is focused on the photodetector (usually photoresistor).

• As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector which unbalances it. The extent of unbalance (which can be related to the sample concentration) is recorded on a strip chart recorder.

The advantages of this type of detector are: (1) Universal response; (2) Low sensitivity to dirt and air bubbles in the

cells; and (3) The ability to cover the entire refractive index

range from 1.000 to 1.750 RI with a single, easily balanced cell.

Refractive index detectors

• It is very sensitive to changes in ambient temp,pressure,flow rate

• It can not be used for the gradient elution technique

• Extremely used for the comp that not adsorb in uv region and not fluorescence

WORKING• It passes the visible light through two compartments• The differential refractometer monitors the deflection of

a light beam caused by the diff in refractive index between the contents of the sample and the reference cell

• The beam of the light from lamp passes through an optical mask

• That confines the beam to the region of the cell • The lens collimates the light beam which passes through

both the cells to a mirror• The mirror reflects the beam back to a lens which focus it

on to a photocell

APPLICATIONS

• It is widely used in the separation and analysis of the polymers

• Used in case of those polymers that contains more than six monomers

• RI is directly proportional to the concentration of the polymer and is practically independent of mol. weight

APPLICATIONS OF HPLC1. Quality control testing of drugs2. In Qualitative & Quantitave analysis3. Therapeutic monitoring of drug metabolism studies4. Separation & control of impurities5. In analysis of biological fluids6. Stability studies7. Study of metabolic pathways in basic biochemical

pathways8. Separation of positional isomers, enantiomers, Optical

isomers9. Industrial applications a. Determination of synthetic intermediates ex: atenolol b. In determining traces of impurity ex:Tolnafate c. Stability studies ex Acyclovir

REFRENCES

• Pharmaceutical Analysis - Dr. A. V. Kasture .• Instrumental Analysis by G .R . Chatwal• Internet source